1. Field of the Invention
This invention relates generally to methods for using hairpin ribozymes to inactivate target RNA molecules. The present invention specifically provides methods for identifying, isolating, and characterizing unknown genes and gene products responsible for detectable phenotypic traits or for characterizing unknown phenotypic effects of known genes, and methods of inactivating target RNAs. Compared to other known ribozymes, the hairpin ribozyme has been discovered to be uniquely effective as a randomized antisense tool.
2. Related Art
A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes. See Castanotto et al. (1994)Advances in Pharmacology 25:289-317 for a general review of the properties of different ribozymes.
The general features of hairpin ribozymes are described e.g., in Hampel et al. (1990) Nucl. Acids Res. 18:299-304; Hampel et al. (1990) European Patent Publication No. 0 360 257; U.S. Pat. No. 5,254,678, issued Oct. 19, 1993; Wong-Staal et al., WO 94/26877; Ojwang et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; Yamada et al. (1994) Human Gene Therapy 1:39-45; Leavitt et al. (1995) Proc. Natl. Acad. Sci. USA 92:699-703; Leavitt et al. (1994) Human Gene Therapy 5:1151-1120; and Yamada et al. (1994) Virology 205:121-126; see FIG. 1. Hairpin ribozymes typically cleave one of two target sequences, NNNNN*GUCNNNNNNNN or NNNNN*GUANNNNNNNN (* denotes the cleavage site, and N can be any nucleotide). See, De Young et al. (1995) Biochemistry 34:15785-15791. The products of the cleavage reaction are a 5xe2x80x2 fragment terminating in a 2xe2x80x2,3xe2x80x2 cyclic phosphate and a 3xe2x80x2 fragment bearing a newly formed 5xe2x80x2-OH. The reaction is reversible; ribozymes also catalyze the formation of phosphodiester bonds. See generally, Buzayan et al. (1986) Nature 323:349-352; Gerlach et al. (1986) Virology 151:172-185; Hampel et al. (1989) Biochemistry 28:4929-4933; Gerlach et al. (1989) Gene 82:43-52; Feldstein et al. (1989) Gene 82:53-61; and Hampel et al. Australian Patent No. AU-B-41594/89.
Ribozymes can be used to engineer RNA molecules prior to reverse transcription and cloning, in a manner similar to the DNA endonuclease xe2x80x9crestrictionxe2x80x9d enzymes. The production of specific ribozymes which target particular sequences is taught in the art (see, e.g., Yu et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344 and Dropulic et al. (1992) J. Virol. 66(3):1432-1441; Wong-Staal et al., WO 94/26877). Ribozymes which cleave or ligate a particular RNA target sequence can be expressed in cells to prevent or promote expression and translation of RNA molecules comprising the target sequence. For instance, expression of hairpin ribozymes which specifically cleave human immunodeficiency (HIV) RNAs prevent replication of the virus in cells. See, Yu et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; Yamada et al. (1994) Virology 205:121-126; Yamada et al. (1994) Gene Therapy 1:38-45; Yu et al. (1995) Virology 206:381-386; Yu et al. (1995) Proc. Nat. Acad. Sci. 92:699-703; and Wong-Staal et al. WO 94/26877 (PCT/US94/05700). The trans-splicing activity of ribozymes can be used to repair defective mRNA transcripts within cells and restore gene expression. Sullenger and Cech (1994) Nature 371:619-622. Quasi-random ribozyme expression vectors were reportedly used to clone target specific ribozymes. Macjak and Draper (1993) J. Cell. Biochem. Supplement 17E, S206:202. A hammerhead ribozyme library comprising a randomized recognition sequence was used for in vitro selection of ribozymes which actively cleave a specific target RNA (Lieber and Strauss (1995) Mol. Cell. Biol. 15:540-551; patent publication 96/01314); ribozymes selected by this method were then expressed in tissue culture cells (id.) and in transgenic mice (Lieber and Kay (1996) J. Virol. 70:3153-3158). In addition, hammerhead ribozyme libraries comprising a randomized catalytic region have been used to select ribozymes that efficiently cleave a specific target RNA. Patent publication WO 92/01806. A library of the ribozyme form of the group I intron of Tetrahymena thermophila having a partially randomized recognition sequence was used for in vitro selection of ribozymes which actively cleave a specific target RNA. Campbell and Cech (1995) RNA 1:598-609.
However, even when both the sequence of the cleavage sites of a specific target RNA and the recognition sequences of ribozymes that cleave that specific RNA are known, targeted cleavage of RNA in vivo has been difficult to achieve (See, e.g., Ojwang et al. (1992) Proc. Natl. Acad. Sci. USA 89:10802-10806), in part for the following reasons: (a) The target site may be hidden within the folds of secondary structure in the substrate RNA, or by interaction with RNA binding molecules. (b) The substrate RNA and the ribozyme may not be present in the same cellular compartment. (c) The ribozyme may be inhibited or inactivated in vivo, either because it is degraded, or because it assumes a secondary structure in vivo that is incompatible with catalytic activity, or because of interactions with cellular molecules. The observed biological effects in some instances can be attributed to simple binding of the ribozyme, as opposed to binding and cleavage. (d) The ribozyme is not produced in sufficient quantities.
The present invention addresses these and other problems.
In the present invention, a hairpin ribozyme gene library having a randomized target recognition sequence, packaged in a vector which is suitable for high level transduction and expression in a wide variety of cells, is used to identify, isolate, and characterize unknown genes and gene products responsible for detectable phenotypic traits, and to characterize unknown phenotypic effects of known genes. In a preferred embodiment, the vector is an adeno-associated virus vector (AAV). The ribozyme gene is preferably operably linked to a transcriptional promoter that allows for optimal inhibition of target RNA expression in vivo, such as a pol III promoter. The result is an unexpectedly high level of expression of the ribozyme gene products. This efficient expression in turn makes possible the in vivo or in vitro selection of ribozyme genes that are active in vivo, even when the target site is not known.
In one embodiment, the invention comprises a method of correlating expression of a nucleic acid that encodes a hairpin ribozyme with the appearance or loss of a detectable phenotype which results from the inhibition or expression of a cellular gene not previously known to result in said phenotype, which involves generating transduced cell clones which express at least one reporter gene or otherwise selectable marker and one or more ribozyme genes from a library of hairpin ribozyme-encoding nucleic acids having randomized target recognition sequences, detecting a phenotypic difference between a transduced cell that expresses said hairpin ribozyme, and a cell of the parental cell line that does not express said hairpin ribozyme, deconvoluting if necessary, isolating and sequencing the ribozyme present in transduced cells that express a selected phenotype. The hairpin ribozyme-encoding nucleic acid is operably linked to an inducible or constitutive promoter. In this and other embodiments, the cells can be eukaryotic, particularly mammalian cells, and the cellular gene can be from the genome of the transformed cell.
In another embodiment, the invention comprises a method of determining unknown phenotypic effects of a coding nucleic acid of known sequence, comprising: simultaneously expressing within a same cell a coding nucleic acid of known sequence and also a hairpin ribozyme that recognizes at least one GUC site present in said coding nucleic acid of known sequence; and then detecting phenotypic differences between cells that simultaneously express said coding nucleic acid of known sequence and also a hairpin ribozyme that recognizes said at least one GUC site present in said coding nucleic acid of known sequence, and cells that express only said coding nucleic acid of known sequence, or cells that express only said hairpin ribozyme that recognizes a GUC site present in said coding nucleic acid of known sequence.
In another embodiment, the invention comprises a method of identifying a nucleic acid whose gene product mediates binding to a selected ligand, comprising transducing a population of parental cells which express a nucleic acid whose gene product mediates binding to a selected ligand with vectors that comprise a library of hairpin ribozyme-encoding nucleic acids having randomized recognition sequences and with a nucleic acid that encodes at least one reporter gene; identifying and cloning a transduced cell that does not bind to said selected ligand, to yield a population of cloned cells that do not bind to said selected ligand; isolating the ribozyme that is expressed in said cloned transduced cells; and determining the sequence of the recognition sequence of the ribozyme; making an oligonucleotide probe consisting of the recognition sequence of the ribozyme; and identifying a nucleic acid whose gene product is recognized by the ribozyme using the oligonucleotide probe. The selected ligand may be one that binds to a cell surface receptor. The ligand may be present on a viral particle, a growth factor, a differentiation factor, any protein with an antibody directed against it. It can be identified by FACS or affinity separation techniques. The ligand may belong to the group consisting of hormone receptors, receptors for molecules that induce apoptosis, and drug receptors.
In another embodiment, the invention comprises a method of identifying regulatory gene products and genes that control the expression of a particular selected nucleic acid, by genetically engineering a population of cells to express in every cell a selected gene operably linked to one or more reporter genes in a vector and at least one member of a library of hairpin ribozyme-encoding nucleic acids having randomized recognition sequences; isolating and cloning a genetically engineered cell wherein the level of expression of the reporter gene is measurably different from that of cells that express the reporter gene but do not express a member of said library of hairpin ribozyme-encoding nucleic acids; and identifying a nucleic acid whose gene product is recognized by a ribozyme expressed in the cloned genetically engineered cells.
In yet another embodiment, the invention comprises a method of identifying a gene whose gene product confers sensitivity to a selected chemical compound or sensitivity to a cytolytic virus or any other microbial entity, comprising transducing a population of parental cells which are sensitive to a selected chemical compound or virus or microbe with vectors that comprise a library of hairpin ribozyme-encoding nucleic acids having randomized recognition sequences; identifying and cloning a transduced cell that is resistant to said selected chemical compound or virus or microbe, to yield a population of cloned transduced cells that are resistant to said selected chemical compound or virus or microbe; and identifying a nucleic acid whose gene product is recognized by a ribozyme expressed by the cloned transduced cells of step b.
In yet another embodiment, the invention comprises a method of identifying a gene whose gene product confers sensitivity to a non-cytolytic virus, comprising transducing a population of parental cells which are sensitive to a selected virus with vectors that comprise a library of hairpin ribozyme-encoding nucleic acids having randomized recognition sequences; identifying and cloning a transduced cell that is resistant to said virus. Resistant cells can be identified by sorting for those cells that lack expression of a viral antigen (ideally a cell surface associated antigen) following infection. Alternatively, cells expressing ribozymes can be selected based on their ability to inactivate expression of one or more viral promoters through the inactivation of viral or cellular genes necessary for transactivation or transexpression of the viral promoter, by linking a selectable marker to the viral promoter and selecting for the loss of reporter gene function.
In yet another embodiment, the invention comprises the generation of a target nucleic acid and an in vitro method of detecting a ribozyme that cleaves said target, comprising hybridizing a library of hairpin ribozyme-encoding nucleic acids having randomized recognition sequences in vitro to a target nucleic acid (RNA or DNA that has been converted to RNA) under stringent hybridization conditions in a solution that does not permit cleavage, wherein the ribozymes having recognition sequences that are complementary to the target nucleic acid hybridize to the recognition site on the target nucleic acid but do not cleave the target nucleic acid; and collecting one or more ribozymes that bind to the target nucleic acid. The target nucleic acid is preferably attached to a solid substrate and consists of RNA or DNA that has been converted to RNA, and can be derived from an isolated chromosome, an isolated nucleic acid that encodes a desired gene product, a selected isolated nucleic acid fragment; an isolated polycistronic nucleic acid; a cDNA library, and a total messenger RNA fraction of a cell. Furthermore, ribozymes with activity against said target can be amplified and the selection procedure can be repeated multiple times. Alternatively, the target nucleic acid is not attached to a solid support and the trans-ligation properties of the hairpin ribozyme are employed to xe2x80x9ctagxe2x80x9d the cleavage products. This ribozyme xe2x80x9ctagxe2x80x9d is then used to amplify the specific active ribozymes.
In a further embodiment, the invention comprises a method for identifying differentially-expressed genes between two cell types, using in vitro selection techniques that are technically easier than currently available methods. Further, the invention comprises identifying a ribozyme or ribozymes that will inactivate the differentially-expressed gene(s).
In another embodiment, the invention comprises an in vivo method of selecting at least one hairpin ribozyme gene that cleaves a target recognition site in a target nucleic acid, comprising generating cells that co-express a library of hairpin ribozyme-encoding nucleic acids having randomized recognition sequences and a nucleic acid that encodes at least one FACS-sortable or otherwise selectable reporter gene, wherein the gene product of at least one ribozyme-encoding nucleic acid cleaves a target sequence in a selected target nucleic acid; isolating and packaging the ribozyme-encoding nucleic acids of said cloned cells; then generating and cloning transduced cells that express at least one ribozyme-encoding nucleic acid whose gene product cleaves a target sequence; and isolating the ribozyme-encoding nucleic acid from the cloned cells.
In another embodiment, the invention comprises a method for identifying genes involved in cancer formation, such as oncogenes or tumor suppressors. This comprises transducing parental cells with vectors that comprise a library of hairpin ribozyme-encoding nucleic acids having randomized recognition sequences; identifying and cloning a transduced cell that has either: a) in the case of oncogenes, lost its transforming potential in tissue culture and nude mice or b) gained transformation capabilities in tissue culture and nude mice, as would be the case for loss of tumor suppressor function.
In another embodiment, the invention comprises a method of detecting a ribozyme that produces a detectable phenotype in a mammal, comprising generating transgenic or chimeric non-human mammals that express nucleic acids that encode hairpin ribozymes that recognize a selected target nucleic acid; screening transgenic mammals for a selected phenotype; and isolating and characterizing the ribozyme-encoding nucleic acids from the cells of the transgenic or chimeric animal. Preferably, the phenotype is a neurological disorder, such as Alzheimer""s disease (Games et al. (1995) Nature 373:523; Moran et al. (1995) PNAS USA 92:5341).
In a separate embodiment, the invention comprises a target-specific ribozyme gene library, generated by collecting ribozymes that bind to and cleave a specific target sequence.
In another embodiment, the invention comprises a differential cell ribozyme gene library, composed of ribozymes that bind to a first cell line but not to a second cell line.
In yet another embodiment, the invention comprises a kit which includes a hairpin ribozyme library having a randomized recognition sequence, packaged in a vector which is suitable for high level expression in a wide variety of cells. The preferred vector is an adenoviral associated vector, and the ribozyme gene is preferably operably linked to a pol III promoter. The kit further comprises reagents and detailed instruction for use in the above methods of the invention.